Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage

Pham, Hang; Nguyen, Tram Thi Ngoc; Nguyen, Lap P.; Han, Sangsoo; Lim, Yun-Sook; Hwang, Soon B.

doi:10.1128/jvi.01240-18

Cited by 13 publications

(10 citation statements)

References 65 publications

(58 reference statements)

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“…4A). Recently, HCV NS5A protein was reported to contribute to increased cellular sensitivity to DNA damage agents (36,43). An increased level of ␥H2AX foci was detected in…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus NS3 Protein Plays a Dual Role in WRN-Mediated Repair of Nonhomologous End Joining

Chen

Hsu

Chou

et al. 2019

J Virol

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Hepatitis C virus (HCV) NS3 protein possesses protease and helicase activities and is considered an oncoprotein in virus-derived hepatocellular carcinoma. The NS3-associated oncogenesis has been studied but not fully understood. In this study, we have identified novel interactions of the NS3 protein with DNA repair factors, Werner syndrome protein (WRN) and Ku70, in both an HCV subgenomic replicon system and Huh7 cells expressing NS3. HCV NS3 protein inhibits WRN-mediated DNA repair and reduces the repair efficiency of nonhomologous end joining. It interferes with Ku70 recruitment to the double-strand break sites and alters the nuclear distribution of WRN-Ku repair complex. In addition, WRN is a substrate of the NS3/4A protease; the level of WRN protein is regulated by both the proteasome degradation pathway and HCV NS3/4A protease activity. The dual role of HCV NS3 and NS3/4A proteins in regulating the function and expression level of the WRN protein intensifies the effect of impairment on DNA repair. This may lead to an accumulation of DNA mutations and genome instability and, eventually, tumor development. IMPORTANCE HCV infection is a worldwide problem of public health and a major contributor to hepatocellular carcinoma. The single-stranded RNA virus with RNA-dependent RNA polymerase experiences a high error rate and develops strategies to escape the immune system and hepatocarcinogenesis. Studies have revealed the involvement of HCV proteins in the impairment of DNA repair. The present study aimed to further elucidate mechanisms by which the viral NS3 protein impairs the repair of DNA damage. Our results clearly indicate that HCV NS3/4A protease targets WRN for degradation, and, at the same time, diminishes the repair efficiency of nonhomologous end joining by interfering with the recruitment of Ku protein to the DNA double-strand break sites. The study describes a novel mechanism by which the NS3 protein influences DNA repair and provides new insight into the molecular mechanism of HCV pathogenesis.

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“…4A). Recently, HCV NS5A protein was reported to contribute to increased cellular sensitivity to DNA damage agents (36,43). An increased level of ␥H2AX foci was detected in…”

Section: Discussionmentioning

confidence: 99%

Hepatitis C Virus NS3 Protein Plays a Dual Role in WRN-Mediated Repair of Nonhomologous End Joining

Chen

Hsu

Chou

et al. 2019

J Virol

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“…Cells seeded on a 24-well plate were transfected with the indicated siRNAs. At the indicated time points, cell viability was measured by using water-soluble tetrazolium salt (WST) (Dail Lab) as we reported previously (5,55).…”

Section: Methodsmentioning

confidence: 99%

PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly

Nguyen

Tran

Suetsugu

et al. 2020

J Virol

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Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. Using protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family, which is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and the Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCα)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCα interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the small interfering RNA (siRNA)-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core protein (core) interaction. We further showed that inhibition of PKCα increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly. IMPORTANCE PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in the virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed a negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCα from binding to PACSIN2. Therefore, the phosphorylation level of PACSIN2 is decreased in HCV-infected cells. We showed that HCV NS5A interrupted PKCα-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.

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“…It has been proposed that the release of XLF from this repair complex promotes subsequent XLF degradation [ 86 ] or alternatively facilitates DNA repair dynamics [ 67 ]. UBE2S is another nuclear target protein of Akt with a suggested role in NHEJ: Akt-dependent phosphorylation of UBE2S at Thr152 protected the protein from proteasomal degradation [ 87 ], thereby presumably increasing resistance to DNA damage [ 88 ]. Herein, Hu and colleagues suggested a role of UBE2S in regulating NHEJ through binding to Ku70, whereas Paul and colleagues proposed a role of UBE2S in RNF8-mediated transcriptional silencing near DNA damage sites [ 87 ], [ 89 ].…”

Section: Role Of Akt In Dna Damage Response and Dsb Repairmentioning

confidence: 99%

“…Taken together, Akt-dependent regulation of its nuclear target proteins mostly improves DNA repair and can thereby promote resistance to genotoxic therapies [ 51 , 56 , 88 , 93 , 94 , 95 , 98 ]. Furthermore, Akt-dependent regulation of cell survival, growth and proliferation can indirectly influence the radiation response to promote therapy resistance [ 51 , 52 , 99 ].…”

Section: Role Of Akt In Dna Damage Response and Dsb Repairmentioning

confidence: 99%

A New Twist in Protein Kinase B/Akt Signaling: Role of Altered Cancer Cell Metabolism in Akt-Mediated Therapy Resistance

Götting

Jendrossek

Matschke

2020

IJMS

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Cancer resistance to chemotherapy, radiotherapy and molecular-targeted agents is a major obstacle to successful cancer therapy. Herein, aberrant activation of the phosphatidyl-inositol-3-kinase (PI3K)/protein kinase B (Akt) pathway is one of the most frequently deregulated pathways in cancer cells and has been associated with multiple aspects of therapy resistance. These include, for example, survival under stress conditions, apoptosis resistance, activation of the cellular response to DNA damage and repair of radiation-induced or chemotherapy-induced DNA damage, particularly DNA double strand breaks (DSB). One further important, yet not much investigated aspect of Akt-dependent signaling is the regulation of cell metabolism. In fact, many Akt target proteins are part of or involved in the regulation of metabolic pathways. Furthermore, recent studies revealed the importance of certain metabolites for protection against therapy-induced cell stress and the repair of therapy-induced DNA damage. Thus far, the likely interaction between deregulated activation of Akt, altered cancer metabolism and therapy resistance is not yet well understood. The present review describes the documented interactions between Akt, its target proteins and cancer cell metabolism, focusing on antioxidant defense and DSB repair. Furthermore, the review highlights potential connections between deregulated Akt, cancer cell metabolism and therapy resistance of cancer cells through altered DSB repair and discusses potential resulting therapeutic implications.

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Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage

Cited by 13 publications

References 65 publications

Hepatitis C Virus NS3 Protein Plays a Dual Role in WRN-Mediated Repair of Nonhomologous End Joining

Hepatitis C Virus NS3 Protein Plays a Dual Role in WRN-Mediated Repair of Nonhomologous End Joining

PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly

A New Twist in Protein Kinase B/Akt Signaling: Role of Altered Cancer Cell Metabolism in Akt-Mediated Therapy Resistance

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